1. Field of the Invention
This invention is in the field of gene therapy, more particularly the field of using neuronal cells to treat brain tumors. The present invention further relates to the field of genetic engineering and medical treatment with genetically engineered stem cells. More particularly, the invention relates to a method of treatment of CNS tumors using genetically engineered neural stem cells (NSCs).
2. Technical Background
An effective gene therapy for the treatment of brain tumors has been an elusive goal for many years. Glioblastoma multiforma, which is virtually untreatable, and the less malignant anaplastic astrocytoma account for about one-quarter of the 5,000 intracranial gliomas diagnosed yearly in the United States; 75 percent of gliomas in adults are of this category. Because of its profound and uniform morbidity, it contributes more to the cost of cancer on a per capita basis than does any other tumor. The patient, commonly stricken in the fifth decade of life, enters a cycle of repetitive hospitalizations and operations while experiencing the progressive complications associated with relatively ineffective treatments of radiation and chemotherapy [“Harrison's Principles of Internal Medicine,” edited by Issetbacher, Braunwald, Wilson, Martin, Fauci and Kasper, 13th Edition, p. 2262, McGraw-Hill, Inc. 1994].
One of the impediments to gene therapy of brain tumors such as gliomas, has been the degree to which they expand, migrate widely and infiltrate normal tissue. Most gene therapy strategies to date are viral vector-based, yet extensive distributions of sufficient amounts of viral vector-mediated genes to large regions and numbers of cells typically in need has often been disappointingly limited. Interestingly, one of the defining features of normal neural progenitors and stem cells is their migratory quality. Neural stem cells (NSCs) are immature, uncommitted cells that exist in the developing, and even adult, CNS and postulated to give rise to the array of more specialized cells of the CNS. They are operationally defined by their ability to self-renew and to differentiate into cells of most (if not all) neuronal and glial lineages in multiple anatomical & development contexts, and to populate developing and/or degenerating CNS regions [Ciage et al., Ann Rev Neurosci 18: 159-92, 1995; Whittemore et al., Molecular Neurobiology 12:13-39 1996; McKay Science 276: 66-71, 1997; Gage F H, Christen Y. (eds.), Research & Perspecti'ves in Nourotciences: Isolation, Characterization, & Utilization of CNS Stem Cells, Springer-Verlag, Heidelberg, Berlin, 1997; Snyder, The Neuroscientist 4, 408-25, 1998].
With the first recognition that neural cells with stem cell properties, reproduced in culture, could be reimplanted into mammalian brain where they could reintegrate appropriately and seamlessly in the neural architecture and stably express foreign genes gene therapists began to speculate how such a phenomenon might be harnessed for therapeutic purposes [Snyder et al., Cell 68: 33-51 1992; Renfranz et al., Cell 66: 713-729, 1991]. These, and the studies which they spawned, provided hope that the use of neural progenitor/stem cells, by virtue of their inherent biology, might circumvent some of the present limitations of presently available gene transfer vehicles (e.g., non-neural cells, viral vectors, synthetic pumps), and provide the basis for a variety of novel therapeutic strategies [for review, see e.g., [Ciage et al., Ann Rev Neurosci 18: 159-92, 1995; Whittemore et al., Molecular Neurobiology 12:13-39 1996; McKay Science 276: 66-71, 1997; Gage F H, Christen Y. (eds.), Research & Perspecti'ves in Nourotciences: Isolation, Characterization, & Utilization of CNS Stem Cells, Springer-Verlag, Heidelberg, Berlin, 1997; Snyder, The Neuroscientist 4: 408-25, 1998; Snyder et al., Current Opin in Pediatrics 8: 558-568, 1996].
The use of neural stem cells as graft material has been clearly illustrated by the prototypical neural progenitor clone, C17.2, a clone with which we have had extensive experience which was used in the studies presented here [Snyder et al., Cell 68: 33-51 1992; Snyder et al., Nature 374: 367-370, 1995; Park, J Neurotrauma 16: 675-87, 1999; Aboody-Guterman et al., NeuroReport 8: 3801-08, 1997]. C17.2 is a mouse cell line from postnatal day 0 cerebellum immortalized by infection with a retroviral construct containing the avian myc gene. This line has been transduced to constitutively express the lacZ and neoR genes. When transplanted into germinal zones throughout the brain, these cells have been shown to migrate, cease dividing, and participate in the normal development of multiple regions at multiple stages (fetus to adult) along the murine neuraxis, differentiating appropriately into diverse neuronal and glial cell types as normal, nontumorigenic cytoarchitectural constituents. They intermingle non-disruptively with endogenous neural progenitor/stem cells, responding to the same spatial and temporal cues in a similar manner. Crucial for therapeutic considerations, the structures to which C17.2 cells contribute develop and maintain neuroanatomical normality. In their earliest therapeutic use, they served to deliver a missing gene product throughout the brains of mice with a lysosomal deficiency state and cross-corrected host cells by release and uptake of a lysosomal enzyme [Snyder et al., Nature 374: 367-370, 1995]. The feasibility of a neural progenitor/stem cell-based strategy for the delivery of therapeutic molecules directly to and throughout the CNS was first affirmed by correcting the widespread neuropathology of a murine model of the genetic neurodegenerative lysosomal storage disease mucopolysaccaridosis type VII, caused by an inherited deletion of the β-glucuronidase (GUSB) gene, a condition that causes mental retardation and early death in humans. Exploiting their ability to engraft diffusely and become integral members of structures throughout the host CNS, GUSB-secreting NSCs were introduced at birth into subventricular germinal zone, and provided correction of lysosomal storage in neurons and glia throughout mutant brains. In so doing, it established that neural transplantation of neural progenitor cells could provide a novel therapeutic modality.
What is needed is a way to treat tumors which are diffuse, infiltrating and/or metastasizing. What is needed is a way to treat tumors locally to maximize the impact on the tumor and reduce the toxicity to the patient.